Non-or minimally invasive monitoring methods using particle delivery methods

ABSTRACT

Methods for sampling an analyte present in a biological system are provided. The methods entail use of particle delivery methods to obtain a sample of an analyte of interest from the system.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. provisional application Ser.No. 60/099,157, filed Sep. 4, 1998, from which priority is claimedpursuant to 35 U.S.C. §119(e)(1) and which application is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

[0002] This invention relates generally to methods of monitoring thepresence and/or concentration of target analytes in an aqueousbiological system. More particularly, the invention relates to methodsfor determining the presence, for example measuring the concentration,of one or more analytes in a transdermally extracted sample. Oneimportant application of the invention involves a sampling method formonitoring blood glucose using non-invasive or minimally invasivesampling techniques.

BACKGROUND

[0003] A number of tests are routinely performed on humans to evaluatethe amount or existence of substances present in blood or other bodyfluids. These tests typically rely on physiological fluid samplesremoved from a subject, either using a syringe or by pricking the skin.One particular test entails self-monitoring of blood glucose levels bydiabetics.

[0004] Diabetes is a major health concern, and treatment of the moresevere form of the condition, Type I (insulin-dependent) diabetes,requires one or more insulin injections per day. Insulin controlsutilization of glucose or sugar in the blood and prevents hyperglycemiawhich, if left uncorrected, can lead to ketosis. On the other hand,improper administration of insulin therapy can result in hypoglycemicepisodes, which can cause coma and death. Hyperglycemia in diabetics hasbeen correlated with several long-term effects, such as heart disease,atherosclerosis, blindness, stroke, hypertension and kidney failure.

[0005] The value of frequent monitoring of blood glucose as a means toavoid or at least minimize the complications of Type I diabetes is wellestablished. According to the National Institutes of Health, glucosemonitoring is recommended 4-6 times a day. Patients with Type II(non-insulin-dependent) diabetes can also benefit from blood glucosemonitoring in the control of their condition by way of diet andexercise.

[0006] Conventional blood glucose monitoring methods generally requirethe drawing of a blood sample (e.g., by finger prick) for each test, anda determination of the glucose level using an instrument that readsglucose concentrations by electrochemical or colorimetric methods. TypeI diabetics must obtain several finger prick blood glucose measurementseach day in order to maintain tight glycemic control. However, the painand inconvenience associated with this blood sampling, along with thefear of hypoglycemia, has lead to poor patient compliance, despitestrong evidence that tight control dramatically reduces long-termdiabetic complications. In fact, these considerations can often lead toan abatement of the monitoring process by the diabetic.

SUMMARY OF THE INVENTION

[0007] The present invention provides a method for sampling an analytepresent in a biological system. More especially, the invention providesa method for sampling an analyte present beneath a target skin ormucosal surface of an individual, said method comprising:

[0008] (a) accelerating particles into and/or across said targetsurface, wherein acceleration of said particles into or across thetarget surface is effective to allow passage of a fluid sample frombeneath the target surface to the target surface; and

[0009] (b) determining the presence of said analyte in said fluidsample.

[0010] The invention also provides use of an inert material for themanufacture of a particulate composition for sampling an analyte presentbeneath a target skin or mucosal surface of an individual by such amethod. The method can be used to determine, for example qualitativelyor quantitatively, the presence of an analyte of interest in thebiological system. The method can also be used to determine the amountor concentration of the analyte of interest. In addition, the method canbe used to continually or continuously measure the concentration of theanalyte.

[0011] The method entails accelerating particles into and/or across atarget surface of the biological system such that the particles allow aquantity of an analyte (e.g., a fluid sample containing or suspected ofcontaining an analyte of interest) to pass from beneath the targetsurface. The analyte can then be contacted with a sensing apparatus toderive a raw detectable signal therefrom, wherein the raw signal iseither indicative of the presence of the analyte, or related to theanalyte concentration. If desired, the analyte can be collected from thetarget surface prior to contact with the sensing apparatus.

[0012] Sampling is carried out such that the analyte of interest istransdermally extracted from the biological system. In this regard, theterms “transdermal extraction” and “transdermally extracted” intend anynon-invasive, or at least minimally invasive method of using particledelivery techniques to facilitate extraction of an analyte from beneatha tissue surface, across skin or mucosal tissue for subsequent analysison, or collection and analysis from the surface thereof. The termsfurther include any such extraction whether or not coupled withapplication of skin penetration enhancers, negative pressure (vacuum orsuction), or other extraction technique.

[0013] Analyte (generally within a volume of fluid) which is extractedfrom the biological system is then either contacted directly with asensing apparatus for obtaining a raw signal indicative of the presenceand/or concentration of the analyte of interest, or collected and thencontacted with the sensing apparatus. This raw signal can be obtainedusing any suitable sensing methodology including, for example, methodswhich rely on direct contact of a sensing apparatus with the biologicalsystem, methods which rely on contact with a collected amount of theextracted analyte, and the like. The sensing apparatus used with any ofthe above-noted methods can employ any suitable sensing element toprovide the raw signal including, but not limited to, physical,chemical, biochemical (e.g., enzymatic, immunological, or the like),electrochemical, photochemical, spectrophotometric, polarimetric,calorimetric, radiometric, or like elements. In preferred embodiments ofthe invention, a biosensor is used which comprises an electrochemicalsensing element.

[0014] The analyte can be any specific substance or component that oneis desirous of detecting and/or measuring in a chemical, physical,enzymatic, or optical analysis. Such analytes include, but are notlimited to, toxins, contaminants, amino acids, enzyme substrates orproducts indicating a disease state or condition, other markers ofdisease states or conditions, drugs of recreation and/or abuse,performance-enhancing agents, therapeutic and/or pharmacologic agents,electrolytes, physiological analytes of interest (e.g., calcium,potassium, sodium, chloride, bicarbonate (CO₂), glucose, urea (bloodurea nitrogen), lactate, and hemoglobin), lipids, and the like. Inpreferred embodiments, the analyte is a physiological analyte ofinterest, for example glucose, or a chemical that has a physiologicalaction, for example a drug or pharmacological agent. As will beunderstood by the ordinarily skilled artisan upon reading the presentspecification, there are a large number of analytes that can be sampledusing the present methods.

[0015] Accordingly, it is a primary object of the invention to provide amethod for sampling an analyte present in a biological system. Theanalyte is typically present beneath a target skin or mucosal surface ofan individual. The method entails the steps of accelerating samplingparticles into and/or across a target surface. Acceleration of thesampling particles into or across the target surface is effective toallow passage of a quantity of the analyte (typically a fluid samplecomprising the analyte) from beneath the target surface to the targetsurface. The sample can contain a diagnostic quantity of the analyte.The presence and/or amount or concentration of the analyte which is soextracted is then determined by direct contact with a sensing apparatus,or the analyte is collected from the target surface and then contactedwith a sensing apparatus.

[0016] An advantage of the invention is that the sampling process can bereadily practiced inside and outside of the clinical setting and withoutpain.

[0017] These and other objects, aspects, embodiments and advantages ofthe present invention will readily occur to those of ordinary skill inthe art in view of the disclosure herein.

DESCRIPTION OF THE FIGURES

[0018]FIG. 1 is a schematic diagram of the modified Franz cell used inExample 1 for in vitro glucose measurement.

[0019]FIG. 2 is a plot of optical density of glucose solution in donorcompartment and glucose extracted from three different skins in the invitro study of Example 1.

[0020] FIGS. 3 to 5 are glucose tolerance test profiles on threeindividual subjects from the in vivo study of Example 2.

[0021]FIG. 6 is a plot of optical density over venous glucose value fromthe study of Example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified analytes or process parameters as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

[0023] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0024] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “a particle” includes a mixture of two or moresuch particles, reference to “an analyte” includes mixtures of two ormore such analytes, and the like.

[0025] A. Definitions

[0026] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although a number ofmethods and materials similar or equivalent to those described hereincan be used in the practice of the present invention, the preferredmaterials and methods are described herein.

[0027] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0028] The term “analyte” is used herein in its broadest sense to denoteany specific substance or component that is being detected and/ormeasured in a physical, chemical, biochemical, electrochemical,photochemical, spectrophotometric, polarimetric, calorimetric, orradiometric analysis. A detectable signal can be obtained, eitherdirectly or indirectly, from such a material. In preferred embodiments,the analyte is a physiological analyte of interest (e.g., aphysiologically active material), for example glucose, or a chemicalthat has a physiological action, for example a drug or pharmacologicalagent.

[0029] As used herein, the term “pharmacological agent” intends anycompound or composition of matter which, when administered to anorganism (human or animal), induces a desired pharmacologic and/orphysiologic effect by local and/or systemic action.

[0030] As used herein, the term “sampling” means extraction of asubstance from any biological system across a membrane, generally acrossskin or tissue. The membrane can be natural or artificial, and isgenerally animal in nature, such as natural or artificial skin, bloodvessel tissue, intestinal tissue, and the like. A “biological system”thus includes both living and artificially maintained systems.

[0031] The term “collection reservoir” is used to describe any suitablecontainment means for containing a sample extracted from an individualusing the methods of the present invention. Suitable collectionreservoirs include, but are not limited to, pads, membranes, dipsticks,swabs, tubes, vials, cuvettes, capillary collection devices, andminiaturized etched, ablated or molded flow paths.

[0032] The terms “sensing device” or “sensing apparatus” encompass anydevice that can be used to measure the concentration of an analyte ofinterest. Preferred sensing devices for detecting blood analytesgenerally include electrochemical devices and chemical devices. Examplesof electrochemical devices include the Clark electrode system (see,e.g., Updike et al. (1967) Nature 214:986-988), and other amperometric,coulometric, or potentiometric electrochemical devices. Examples ofchemical devices include conventional enzyme-based reactions as used inthe Lifescan® glucose monitor (see, e.g., U.S. Pat. No. 4,935,346 toPhillips et al.). Detection and/or quantitization of a chemical signalcan also be carried out using readily available spectrophotometricmonitoring devices.

[0033] The term “individual” encompasses any warm-blooded animal,particularly including a member of the class Mammalia such as, withoutlimitation, humans and nonhuman primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

[0034] B. General Methods

[0035] The invention relates to a method for sampling analytes presentin a biological system, typically a physiologically active material thatis present beneath a target skin or mucosal surface of an individual.The method entails two general steps, a sampling step and adetermination step. The sampling step can be generalized as follows.Small sampling particles are accelerated into and/or across a targetsurface. Acceleration and penetration of these particles is sufficientto create passages which allow a quantity of an analyte of interest toflow, exude or otherwise pass from beneath the target surface to thetarget surface. The target surface generally has an overall size rangingfrom about 0.1 to about 5 cm².

[0036] The sampling particles typically comprise an inert material. Thematerial may be dissolvable such as commonly employed physiologicallyacceptable soluble materials including sugars (e.g., mannitol, sucrose,lactose, trehalose, and the like) and soluble or dissolvable polymers.Alternatively, the sampling particles can be comprised of insolublematerials such as starch, TiO₂, calcium carbonate, phosphate salts,hydroxy apatite, or even polymers or metals such as gold, platinum ortungsten. Insoluble materials are sloughed off with the normal skin ormucosal tissue renewal process. Preferred materials are lactose, lacticacid, mannitol and polyethylene glycol such as PEG 8000.

[0037] If desired, the sampling particles can be coated with a locallyactive agent which facilitates the sampling step. For example, thesampling particles can be coated with a pharmacological agent such as avasoactive agent or an anti-inflammatory agent. The vasoactive agent isgenerally used to provide short-acting vasoactivity in order to maximizefluid access (maximize the analyte sample), whereas theanti-inflammatory agent is generally used to provide localanti-inflammatory action to protect the target site. The samplingparticles can also be coated with an osmotically active agent tofacilitate the sampling process.

[0038] The sampling particles can be delivered from a needleless syringesystem such as those described in commonly owned InternationalPublication Nos. WO 94/24263, WO 96/04947, WO 96/12513, and WO 96/20022,all of which are incorporated herein by reference. Delivery of samplingparticles from these needleless syringe systems is generally practicedwith particles having an approximate size generally ranging from 0.1 to250 μm, preferably ranging from about 10-70 μm. Particles larger thanabout 250 μm can also be delivered from the devices, with the upperlimitation being the point at which the size of the particles wouldcause untoward pain and/or damage to the tissue.

[0039] The actual distance which the delivered particles will penetratea target surface depends upon particle size (e.g., the nominal particlediameter assuming a roughly spherical particle geometry), particledensity, the initial velocity at which the particle impacts the surface,and the density and kinematic viscosity of the targeted skin tissue. Inthis regard, optimal particle densities for use in needleless injectiongenerally range between about 0.1 and 25 g/cm³, preferably between about0.9 and 1.5 g/cm³, and injection velocities generally range betweenabout 100 and 3,000 m/sec. With appropriate gas pressure, particleshaving an average diameter of 10-70 μm can be readily acceleratedthrough the nozzle at velocities approaching the supersonic speeds of adriving gas flow. Preferably, the pressure used when accelerating theparticles will be less than 30 bar, preferably less than 25 bar and mostpreferably 20 bar or less.

[0040] Alternatively, the sampling particles can be delivered from aparticle-mediated delivery device such as a so-called “gene-gun” typedevice which delivers particles using either a gaseous or electricdischarge. An example of a gaseous discharge device is described in U.S.Pat. No. 5,204,253. An explosive-type device is described in U.S. Pat.No. 4,945,050. One example of a helium discharge-type particleacceleration apparatus is the PowderJect XR® instrument (PowderJectVaccines, Inc., Madison), Wis., which instrument is described in U.S.Pat. No. 5,120,657. An electric discharge apparatus suitable for useherein is described in U.S. Pat. No. 5,149,655. The disclosure of all ofthese patents is incorporated herein by reference.

[0041] After the sampling particles have been delivered into the targetsurface, a fluid sample passes to the target surface. Typically this isa sample of, or containing, interstitial fluid. Passage of the fluidsample to the surface may be substantially instantaneous, or may occurover a period of time. The quantity of the sample which is released tothe target surface may be varied by altering conditions such as the sizeand/or density of sampling particles and the settings of the apparatusused to delivery the particles. The quantity of fluid released may oftenbe small, such as <1 μl which is generally sufficient for detection ofthe analyte.

[0042] Once the sample has passed to the target surface, the presenceand/or amount or concentration of the analyte in the sample isdetermined. The sample may be contacted with a suitable sensingapparatus. This detection step can, of course, be carried out in acontinual or continuous manner. Continual or continuous detection allowsfor monitoring of target analyte concentration fluctuations.Furthermore, the sample believed to contain the analyte can first becollected from the target surface prior to being contacted with thesensing apparatus.

[0043] The sample may be collected from the target surface in a numberof ways. For example pads, membrane dipsticks, swabs, tubes, vials,curvettes, capilliary collection devices and miniaturized etched,ablated or molded flow paths may be used as collection reservoirs. In apreferred aspect an absorbent material is passed over the target surfaceto absorb the fluid sample from the target surface for subsequentdetection of the presence or amount of analyte. The absorbent materialmay be in the form of a pad or swab. The absorbent material mayadditionally incorporate means to facilitate detection of the analytesuch as an enzyme as described in more detail below.

[0044] The absorbent material may be applied to the target surface andsubsequently contacted with a detection means to detect the analyte. Theabsorbent material may comprise a hydrogel. Suitable gelling agents forforming a hydrogel include carbopol, calcium lactate, cellulose gum,klucel (HPMC), natrosol, gelatin powder or sodium alginate. The gellingagents may be present in water at levels such as 1% by weight in water.

[0045] The gel may be applied to the target surface and sufficient timeallowed for analyte from the target surface to equilibrate in the gelprior to the detection step. The time may be quite short such as from 30seconds to 5 minutes. Detection may then be carried out by applying thesensing means to the gel such as by contacting a membrane containing asuitable enzyme system for the analyte with the hydrogel.

[0046] The determination step can be generalized as follows. An initialstep can entail obtaining a raw signal from a sensing device, whichsignal is related to a target analyte present in the biological system.The raw signal can then be used directly to obtain an answer about theanalyte, for example a yes or no answer relating to the presence of theanalyte, or a direct measurement indicative of the amount orconcentration of the extracted analyte. The raw signal can also be usedindirectly to obtain information about the analyte. For example, the rawsignal can be subjected to signal processing steps in order to correlatea measurement of the sampled analyte with the concentration of thatanalyte in the biological system. Such correlation methodologies arewell known to those skilled in the art.

[0047] Detection may be carried out by any suitable method which allowsfor detection of a derived analyte. The analysis may be physical,chemical, biochemical, electrochemical, photochemical,spectrophotometric, polermetric, colormetric or radiometric analysis.

[0048] In order to facilitate detection of the analyte, an enzyme may bedisposed on the active surface or portion of a sensing apparatus whichis contacted with the extracted analyte (e.g., an extracted samplecontaining the analyte), or included within one or more collectionreservoirs which are used to collect the extracted analyte. Such enzymesmust be capable of catalyzing a specific reaction with the extractedanalyte (e.g., glucose) to the extent that a product of the reaction canbe sensed (e.g., detected electrochemically from the generation of acurrent which current is detectable and proportional to the amount ofthe analyte which is reacted). A suitable enzyme is glucose oxidasewhich oxidizes glucose to gluconic acid and hydrogen peroxide. Thesubsequent detection of hydrogen peroxide on an appropriate biosensorelectrode generates two electrons per hydrogen peroxide molecule whichcreate a current which can be detected and related to the amount ofglucose entering the device. Glucose oxidase (GOx) is readily availablecommercially and has well known catalytic characteristics. However,other enzymes can also be used, so long as they specifically catalyze areaction with an analyte or substance of interest to generate adetectable product in proportion to the amount of analyte so reacted.

[0049] A number of other analyte-specific enzyme systems can be used inthe methods of the invention. For example, when using a common biosensorelectrode that detects hydrogen peroxide, suitable enzyme systems can beused to detect ethanol (an alcohol oxidase enzyme system), or similarlyuric acid (a urate oxidase system), cholesterol (a cholesterol oxidasesystem), and theophylline (a xanthine oxidase system). Hydrogelscontaining these analyte-specific enzyme systems can be prepared usingreadily available techniques familiar to the ordinarily skilled artisan.

[0050] Preferred sensing devices are patches that include an enzyme orother specific reagent which reacts with the extracted analyte ofinterest to produce a detectable color change or other chemical signal.The color change can be assessed by comparison against a standard todetermine analyte amount, or the color change can be detected usingstandard electronic reflectance measurement instruments. One such systemis transdermal glucose monitoring system available from TechnicalChemicals and Products, Inc (TCPI) of Pompano Beach, Fla. Anothersuitable system is described in U.S. Pat. No. 5,267,152 to Yang et al.(a device and method for measuring blood glucose concentration usingnear-IR radiation diffuse-reflection laser spectroscopy. Similar near-IRspectrometric devices are also described in U.S. Pat. No. 5,086,229 toRosenthal et al. and U.S. Pat. No. 4,975,581 to Robinson et al. U.S.Pat. No. 5,139,023 to Stanley describes a blood glucose monitoringapparatus that relies on a permeability enhancer (e.g., a bile salt) tofacilitate transdermal movement of glucose along a concentrationgradient established between interstitial fluid and a receiving medium.U.S. Pat. No. 5,036,861 to Sembrowich describes a passive glucosemonitor that collects perspiration through a skin patch, where acholinergic agent is used to stimulate perspiration secretion from theeccrine sweat gland. Similar perspiration collection devices aredescribed in U.S. Pat. No. 5,076,273 to Schoendorfer and U.S. Pat. No.5,140,985 to Schroeder. Detection of extracted glucose is carried outusing standard chemical (e.g., enzymatic) colormetric or spectrometrictechniques.

[0051] Alternatively, an iontophoretic transdermal sampling system canbe used in conjunction with the present invention, for example where theinstant particle method is used to pre-treat a skin site to facilitateimproved sampling from a GlucoWatch system (Cygnus, Redwood, Calif.).This iontophoretic system is described in Gliebfeld et al (1989), Pharm.Res. 6(11): 988 et seq and U.S. Pat. No. 5,771,890.

[0052] C. Experimental

[0053] Below are examples of specific embodiments for carrying out themethods of the present invention. The examples are offered forillustrative purposes only, and are not intended to limit the scope ofthe present invention in any way.

[0054] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

EXAMPLE 1

[0055] Introduction

[0056] The interstitial fluid is the clear body fluid between cells onthe top surface layer of skin. The glucose level in this fluid directlyindicates the glucose level in blood. A needleless syringe device cancreate diffusion pathway into these layers and will allow the collectionof a small amount (<1 ml) of interstitial fluid from which the glucoselevel can be measured.

[0057] Materials and Methods

[0058] Lactose monohydrate, NF, grade, was obtained from Amresco®(Solon, Ohio). The lactose powder was sieved to 38-53 μm using U.S.Standard Sieve (Chicago, Ill.). D-(+) glucose was obtained from Sigma(St. Louis, Mo.). Human cadaver skin was supplied from New YorkFirefighter Skin Bank (New York, N.Y.), was pretreated with 80% balancedsalt solution, 10% calf serum and 10% glycerol and frozen at the skinbank. The skin was used as supplied after 1-2 hours thawing at roomtemperature.

[0059] Modified Franz cells (6.9 ml) were designed for investigation ofin vitro glucose extraction (FIG. 1). The donor part in the bottom ofthe cell was filled with different glucose concentrations ranging from10-500 mg/dl. The temperature of the glucose solution was maintained at32° C. during the experiments. Human cadaver skin of a thickness of200-300 mm was used. The skin was punched out using a 1 inch (2.54 cm)die cutter and placed on the modified Franz cell.

[0060] After a few hours for equilibration, 2 mg of lactose particles of38-53 μm were filled into tri-laminate cassette of 20 μm polycarbonatemembrane and were injected onto the skin tissue using a PowderJect ND 1needleless syringe device fitted with a supersonic nozzle. Devicepressure for particle administration was 20 bar. Then, 5 μl of hydrogelwas placed on the top of the injected skin and a One Touch enzymaticmembrane (Lifescan, Milpitas, Calif.) was contacted with the gel for oneminute. The optical density of the membrane was then measured with aDensitometer (Hercules, Calif.).

[0061] Results

[0062] Table 1 is a summary of measured optical density of membranescontacted with the donor part (bottom part of the Franz cell) or thehydrogel at the top of the treated skin. As can be seen, the opticaldensity of extracted glucose from the top of the three different skinsamples is proportionally increased from 0.04-0.08 to 0.65-0.89 as theoptical density of donor part is increased from 0.10 to 0.71. FIG. 2 isthe summary plot of optical density from three skin samples over donorpart. The correlation (r² value) was calculated to be 0.92 over thethree skin samples.

[0063] These in vitro experimental results demonstrate that there isgood correlation (r²=0.92) between the measured values of extractedglucose using the particle delivery methods of the present invention anddonor compartment which simulated human interstitial fluid. TABLE 1Glucose standard solution and optical density of glucose solution indonor compartment and glucose extracted from three different skinsamples Glucose Standard Donor (mg/dl) Compartment Skin 1 Skin 2 Skin 3 10 0.099 0.06  0.04  0.075  50 0.184 0.17  0.19  0.285 100 0.323 0.2650.325 0.26  250 0.56  0.725 0.665 0.54  500 0.721 — 0.895 0.65 

EXAMPLE 2

[0064] Introduction

[0065] The in vitro study with human cadaver skin set out in Example 1demonstrated that there is a solid correlation of “systemic” glucoselevels with fluid samples accessed via a particle delivery method(r²=0.92). Therefore, this study was carried out to compare glucoselevels from two blood sources (venous blood glucose and capillary bloodglucose) with the glucose level of interstitial fluid accessed via aneedleless injection device in human subjects.

[0066] Additionally, on the needleless injection sites, visual grade oferythema or edema using the Draize scale, change in chromaticity, changein transepidermal water loss (TEWL) and any sensation of pain (VAS) weremeasured to indicate stratum corneum disruption and confirm particlepenetration. Measurements were performed immediately after particleinjection and at 24 and 48 hours post-sampling. These measurementsprofile any correlation between venous glucose, capillary glucose andinterstitial glucose with skin tolerability.

[0067] Materials and Methods

[0068] 120 diabetic subjects were included in the study. The subjectswere male or female Type I or Type II diabetics of ages 18-70 butexcluding diabetics with any skin disorder at the sites of testing,including eczema, psoriasis, etc. or with a history of recurrent skininfections. Sampling sites were antecubital fossa (venous), fingertip(capillary) and volar forearm (particle delivery).

[0069] The needleless syringe particle delivery device and compoundswere as follows:

[0070] Powder: lactose, particle size of 20-38 mm, sieved.

[0071] Cassette: trilaminate with 10 μm polycarbonate membranes(upstream membrane was slit), 1.0 mg powder payload, prepared andpackaged in a clean laboratory environment and terminally sterilisedwith γ radiation.

[0072] Device: A dermal PowderJect™ ND1 needleless syringe device fittedwith a supersonic nozzle, 5 cc expansion chamber modified to accommodatetrilaminate cassettes, 10 bar compressed air pressure, push buttonstainless gas cylinder.

[0073] The needleless syringe device was held as vertically as possiblewith the nozzle placed firmly against the skin, ensuring there was nogap between the skin and the end of the device. The device was actuatedby pressing down the button at the top.

[0074] TEWL and chromaticity were assessed for each volar forearminjection site prior to powder delivery. After the powder was deliveredto the site, TEWL was measured for each injection site. Each subject wasasked to describe the sensation of administration in a few words andrecord the sensation on a 100 mm visual analog scale (VAS) for pain,with 100 being the pain of finger-stick and 0 being no pain.

[0075] A LifeScan™ glucose detection membrane strip was moistened with 5μl of hydrogel and applied to the powder-injected site for 1 minute. Thetreated sites were swabbed and chromaticity measured for comparison withpre-treatment values. Capillary blood samples were collected by fingerstick for glucose analysis using the LifeScan One-Touch system. Bloodsamples were collected from a vein in the antecubital fossa for glucoseanalysis. The colour intensity of the membranes were quantified with theBio-Rad densitometer. TEWL and chromaticity of the injected site weremeasured at 24 and 48 hours post injection.

[0076] The protocol used for an oral glucose tolerance test was asfollows. All drug therapy that could affect the test was discontinuedfor at least 3 days before the test. The subjects were instructed toingest a carbohydrate intake of at least 150 g/day for 3 days before thetest, and fast 14 to 15 hours (from 6 pm of the day preceding the testto 8 to 9 am of the day of the test). Test subjects abstained fromtobacco, coffee, tea, food, and alcohol during the test. The subjectssat quietly upright during the test. Slow walking was permitted butvigorous exercise was avoided. Before a glucose load was given, FastingPlasma Glucose (FPG) was measured via the venous, capillary andinterstitial glucose sampling techniques described above. 75 g ofglucose was given as a 25% solution (400 ml). Samples were collected andthe capillary and interstitial glucose levels were measured as above at30, 60, 90, 120 min.

[0077] Results

[0078] The glucose tolerance test results from three subjects aredepicted in FIG. 3 to FIG. 5. The blood glucose concentration (mg/dl)and optical density from reaction with glucose in interstitial fluid areshown. In order to combine the results into the same Figure, opticaldensity was multiplied by 1000.

[0079] The dynamic glucose range of subject 1 was 150 to 270 mg/dl. Theoptical density according to the blood glucose value correlated wellwith venous glucose concentration (FIG. 3). The dynamic glucose range ofsubject 2 was 160 mg/dl to 400 mg/dl. There was a correlation betweenvenous glucose and optical density except at the one hour time point(FIG. 4). A similar trend could be observed in subject 3 (FIG. 5).

[0080] In FIG. 6, the optical density readings from interstitial fluidglucose were plotted over venous blood glucose of all subjects. Theerror grid method was applied to the collected data and plotted. Mostdata points are located in region A and B which are clinicallycorrelated regions. The r value from a linear regression analysis was0.76. Table 2 reports a comparison of error grid analyses between theinvasive Glucometer, the iontophoretic GlucoWatch method, and theparticle delivery method according to the invention. TABLE 2 Clinicalresult comparision between Glucometer ®, Gluco Watch ® and the inventionZone Glucometer ® Gluco Watch ® Invention A 91.3 73.9 77.3 B 7.9 22.720.8 C 0 0.06 0 D 0.8 3.41 1.9 E 0 0 0

[0081] With respect to pain, most subjects (>90%) preferred the particledelivery method according to the invention over the Glucometer andGlucoWatch glucose measuring methods. However, there was some erythemanoted in some subjects that resolved after 3-4 days with the methodologyof the invention.

[0082] Accordingly, novel monitoring methods are disclosed. Althoughpreferred embodiments of the subject invention have been described insome detail, it is understood that obvious variations can be madewithout departing from the spirit and the scope of the invention asdefined by the appended claims.

What is claimed is:
 1. A method for sampling an analyte present beneatha target skin or mucosal surface of an individual, said methodcomprising: (a) accelerating particles into and/or across said targetsurface, wherein acceleration of said particles into or across thetarget surface is effective to allow passage of a fluid sample frombeneath the target surface to the target surface; and (b) determiningthe presence of said analyte in said fluid sample.
 2. The method ofclaim 1 wherein the analyte is a physiologically active material.
 3. Themethod of claim 1 wherein acceleration of the particles into the targetsurface in step (a) serves to increase the permeability of the targetsurface.
 4. The method of claim 1 wherein the particles are acceleratedtoward the target surface in step (a) using a needleless syringe device.5. The method of claim 4 , wherein the particles are accelerated towardthe target surface at a velocity of about 100 to 2,500 m/sec.
 6. Themethod of claim 1 wherein the particles have a size predominantly in therange of about 0.1 to 250 μm.
 7. The method of claim 1 wherein theparticles are coated with a locally active agent.
 8. The method of claim7 , wherein the particles are coated with an osmotically active agent ora pharmacological agent.
 9. The method of claim 8 , wherein thepharmacological agent is a vasoactive agent or an anti-inflammatoryagent.
 10. The method of claim 1 wherein the target surface in step (a)has an overall size ranging from about 0.1 to about 5.0 cm².
 11. Themethod of claim 1 wherein the fluid sample comprises interstitial fluid.12. The method of claim 1 wherein step (b) comprises collecting a samplefrom the target surface.
 13. The method of claim 12 , wherein thecollection step entails contacting the target surface with an absorbentmaterial.
 14. The method of claim 13 , wherein the absorbent material iscoated with an enzyme that reacts specifically with the analyte toproduce a detectable signal.
 15. The method of claim 14 , wherein thephysiologically active material is glucose and the enzyme is glucoseoxidase.
 16. The method of claim 15 , wherein the detectable signal isquantitative and can be correlated with a blood glucose level in theindividual.
 17. The method of claim 14 , wherein the physiologicallyactive material is ethanol and the enzyme is an alcohol oxidase enzyme.18. The method of claim 17 , wherein the detectable signal isqualitative and is indicative of the presence or absence of ethanol inthe individual or the detectable signal is quantitative and can becorrelated with a blood alcohol level in the individual.
 19. The methodof claim 12 wherein the sample is collected into a collection meanswhich is contacted with the target surface after the particles areaccelerated into said surface.
 20. The method of claim 12 wherein theacceleration and collection steps are repeated at least once over thecourse of a day in order to provide for continual monitoring of thephysiologically active material in the individual.
 21. The method ofclaim 1 wherein step (b) comprises contacting the sample which haspassed to the target surface with a sensing apparatus that detects thepresence or amount of said analyte.
 22. Use of an inert material for themanufacture of a particulate composition for sampling an analyte presentbeneath a target skin or mucosal surface of an individual by a methodcomprising: (a) accelerating particles into and/or across said targetsurface, wherein acceleration of said particles into or across thetarget surface is effective to allow passage of a fluid sample frombeneath the target surface to the target surface; and (b) determiningthe presence of said analyte in said fluid sample.